Mapping human pre-rRNA processing and modification at single nucleotide resolution using long read nanopore sequencing.

Ribosome biogenesis requires the synthesis and sequential processing of precursor rRNAs (pre-rRNAs) into mature rRNAs. Traditional methods such as northern blotting and metabolic labeling provide limited resolution. Here, we present NanoRibolyzer, a nanopore-based long-read sequencing approach that enables ab initio identification and quantification of rRNA precursors while simultaneously mapping RNA modifications. Using supervised and unsupervised mapping, we detect both known and previously uncharacterized pre-rRNAs and delineate cleavage events at single-nucleotide resolution. A simple cell-fractionation protocol further separates nuclear and cytoplasmic pre-rRNAs, allowing spatial deconvolution of processing pathways. By projecting each sequenced molecule in a two-dimensional space using its starting and ending coordinates, we generate an intuitive representation in which the activity of the 5' → 3' and 3' → 5' exoRNases can be tracked as they mature pre-rRNAs one nucleotide at a time. Targeted knockdowns of ribosome-assembly factors quantify accumulation of intermediates and reveal condition-specific processing "fingerprints" with biomarker potential. High-resolution re-analysis of known factors uncovers unexpected functions. Additionally, pseudouridine mapping shows that the primary 47S transcript is extensively modified, whereas aberrant intermediates (34S and 36S-C) are hypomodified. With its high resolution and unique discovery mode, NanoRibolyzer provides new insights into rRNA processing and modification, greatly advancing our understanding of ribosome biogenesis.